US20070140972A1 - Targeting compositions and preparation therof - Google Patents

Targeting compositions and preparation therof Download PDF

Info

Publication number
US20070140972A1
US20070140972A1 US10/576,086 US57608604A US2007140972A1 US 20070140972 A1 US20070140972 A1 US 20070140972A1 US 57608604 A US57608604 A US 57608604A US 2007140972 A1 US2007140972 A1 US 2007140972A1
Authority
US
United States
Prior art keywords
seq
cyclo
ctthwgftlc
grenyhg
peptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10/576,086
Other languages
English (en)
Inventor
Ying Zhu
Heli Valtanen
Sami Kaukinen
Oula Penate Medina
IIkka Simpura
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
CTT Cancer Targeting Technologies Oy
Original Assignee
CTT Cancer Targeting Technologies Oy
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by CTT Cancer Targeting Technologies Oy filed Critical CTT Cancer Targeting Technologies Oy
Assigned to CTT CANCER TARGETING TECHNOLOGIES OY reassignment CTT CANCER TARGETING TECHNOLOGIES OY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: VALTANEN, HELI, KAUKINEN, SAMI, PENATE MEDINA, OULA, SIMPURA, ILKKA, ZHU, YING
Publication of US20070140972A1 publication Critical patent/US20070140972A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • the present invention relates to targeted cancer therapy and tumour imaging, and concerns specifically new derivatives of small matrix metalloproteinase inhibitor peptides.
  • the peptide derivatives obtained have improved properties and may be used in the preparation of targeting compositions together with suitable linker molecules.
  • targeting compositions are useful in therapeutic and imaging liposome compositions for cancer treatment and diagnostics.
  • This shell prevents the adsorption of various plasma proteins (opsonins) to the liposome surface so that liposomes are not recognized and taken up by the reticulo-endothelial system.
  • Enhanced selectivity can be obtained by attaching to the surface of the liposome specific antibodies or small peptides recognizing plasma membrane antigens of the target cell, thus augmenting the uptake of the liposome by the cell (Storm and Crommelin, 1998; Dagar et al, 2001; Pe ⁇ ate Medina et al., 2001).
  • MMPs Matrix metalloproteinases
  • MMP-2 and MMP-9 Elevated or un-regulated expression of gelatinases and other MMPs can contribute to the pathogenesis of several diseases, including tumour angiogenesis and metastasis, rheumatoid arthritis, multiple sclerosis, and periodontitis. Random phage peptide libraries have been screened in order to develop a selective inhibitor against this MMP subgroup.
  • CTT The most active peptide derived, abbreviated CTT, was found to selectively inhibit the activities of MMP-2 and MMP-9 (Koivunen et al., 1999).
  • CTT-displaying phages were accumulated in the tumour vasculature after their intra-venous injection into the recipient mice.
  • Targeting of the phage to tumours was inhibited by the co-administration of the CTT peptide (Koivunen et al., 1999).
  • MMP-2 Toth et al., 1997) and MMP-9 (Brooks et al., 1996) are bound by specific cell surface receptors
  • these enzymes represent potential receptors for liposome targeting to invasive cells, such as tumour cells and angiogenic endothelial cells.
  • CTT peptide By mixing CTT peptide with liposomes, enhanced tumour targeting and uptaking can be achieved (Pe ⁇ ate Medina et al., 2001).
  • CTT2 peptide and its derivatives may be covalently attached to suitable linker molecules, especially synthetic lipids.
  • the peptide/lipid composition is purified by a specific method.
  • the composition forms micelles in aqueous solutions and can be incorporated into liposomes.
  • this invention creates a novel and versatile targeting tool for different types of liposomal formulations of pharmaccuticals and imaging agents. The use of the targeting tool is shown to improve the biodistribution profile and the therapeulical efficacy of the drug formulation.
  • the peptide/lipid composition itself also has tumour imaging function in vivo.
  • Other derivatives of the CTT2 peptide were prepared in order to improve solubility of the peptide and usefulness thereof in tumour imaging.
  • FIG. 1 Thin layer chromatography (TLC) analysis of the coupling reaction. Lane 1, CTT2 peptide control; Lane 2, DSPE-PEG-NHS control; Lane 5, the supernatant after the diethyl ether treatment; Lane 8, the pellet suspension after the diethyl ether treatment.
  • TLC Thin layer chromatography
  • FIG. 2 The result of the HPLC gel filtration to separate the CTT2-PEG-DSPE compound from the CTT2 peptide.
  • the first peak shown in the graph contains the product, CTT2-PEG-DSPE.
  • the last peak shown in the graph contains the CTT2 peptide.
  • FIG. 3 a MALDI-TOF analysis of the CTT2 peptide.
  • FIG. 3 b MALDI-TOF analysis of the DSPE-PEG-NHS.
  • FIG. 3 c MALDI-TOF analysis of the CTT2-PEG-DSPE after the HPLC purification.
  • FIG. 4 Tumour accumulation of CTT2-coated Doxil®/Caelyx® and Doxile/Caelyx® in ovarian cancer xenograft mice over a period of 96 hours.
  • FIG. 5 Survival of tumour-bearing mice after the treatment with different drug/liposoine formulations.
  • FIG. 6 The biodistribution study of 1-125-CTT2-PEG-DSPE.
  • the in vivo biodistribution of the 125 I-labeled micelle was assessed at two time points in NMRI/nude mice carrying human ovarian tumours on their lower back. Results are expressed as percentage of injected dose per 1 g of tissue (% ID/1 g). All values are indicated as mean ⁇ SD of 5 mice.
  • FIG. 7 a Molecular structure of amidated CTT2 peptide.
  • FIG. 7 b Molecular structure of G ⁇ K derivative of the CTT2 pcptide.
  • FIG. 7 c Molecular structure of G ⁇ K(DOTA) derivative of the CTT2 peptide.
  • FIG. 7 d Molecular structure of an indium-labeled G ⁇ K(DOTA)-CTT2 peptide.
  • FIG. 7 e Molecular structure of Ac-CTT2-K-NH 2 peptide.
  • FIG. 7 f Molecular structure of Ac-CTT2-K(DOTA)-NH 2 , peptide.
  • FIG. 7 g Molecular structure of 6F-Trp derivative of the CTT2 peptide.
  • FIG. 7 h Molecular structure of 5F-Trp derivative of CTT2 peptide.
  • FIG. 7 i Molecular structure of 5-OH-Trp derivative of CTT2 peptide.
  • FIG. 8 The biodistribution study of I-125 labelled 6F-Trp CTT2 (GRENYHGCTTH[6-fluoro]WGFTLC)-peptide.
  • the in vivo biodistribution of the 125 I-labeled peptide was assessed at two time points in NMRI/nude mice carrying human ovarian tumours on their lower back. Results are expressed as percentage of injected dose per 1 g tissue (% ID/1 g). All values are indicated as mean ⁇ SD of 5 mice.
  • the invention describes a hydrophilic peptide and its derivatives, which can be used in cancer therapeutics and tumour imaging, as well as a process to synthesize such peptides.
  • the peptide is the cyclic CTT2 peptide having the amino acid sequence GRENYHGCTTHWGFTLC (SEQ ID NO:1), which peptide is used as an efficient targeting tool for a liposomal formulation of pharmaceuticals or imaging agents.
  • the peptide (CTT2) is first covalently attached (coupled) to the end group of the poly(ethylene glycol) polymer chain of the PEG phospholipids, DSPE-PEG.
  • the CTT2-PEG-DSPE suspension which forms micelles in an aqueous solution, is then incorporated to the pre-formed liposomes that are loaded with pharmaceuticals or imaging agents.
  • this invention creates a novel and versatile targeting tool for different types of liposomal formulations of pharmaceuticals and imaging agents.
  • the use of this targeting tool is shown to improve the biodistribution profile and the therapeutical efficacy of the drug formulation. Separating the coupling and the incorporation steps makes the system versatile. The physical stress imposed on the peptide and its bond to the PEG phospholipid by conventional liposome formation procedure is avoided.
  • the invention also describes such derivatives of the CTT2 peptide, which have improved solubility and better suitability in tumour imaging.
  • any peptide having suitable targeting capacity can be attached to a liposome with any composition and loaded with any substances. Consequently, the liposome can carry as a pharmaceutical a chemotherapeutic agent, e.g. doxoribicin, cisplatin or paclitaxel.
  • a chemotherapeutic agent e.g. doxoribicin, cisplatin or paclitaxel.
  • the liposome can also carry an imaging agent.
  • the peptides can be attached to suitable able nanoparticles as well.
  • Useful peptides having suitable targeting capacity include for instance the matrix metalloproteinase inhibitory peptides described in the international patent applications WO 99/47550 and WO 02/072618.
  • amidated form of the CTT2 peptide i.e. GRENYHG-cyclo-(CTTHWGFTLC)-NH 2
  • the new derivatives thereof described herein i.e. the peptides KRENYHG-cyclo-(CTTHWGFTLC), K(DOTA)RENYHG-cyclo-(CTTHWGFTLC), K(DOTA(ln))-RENYHG-cyclo-(CTTHWGFTLC), Ac-GRENYHG-cyclo-(CTTHWGFTLC)K-NH 2 , Ac-GRENYHG-cyclo-(CTTHWGFTLC)K(DOTA)-NH 2 , GRENYHG-Cyclo(CTTH(d,1-6-Fluoro-W)GFTLC)-NH 2 , GRENYHG-Cyclo(CTTH(d,1-5-Fluoro-W)GFTLC)-NH 2 and GRENYHG-Cyclo-(CTTH(d,l-5-OH-W
  • a general object of the present invention is a targeting composition, which comprises a peptide having tumour-targeting capacity, preferably one of the above-indicated peptides, attached to a suitable lipid.
  • the composition obtained can be used as a targeting moiety in various medical and diagnostic applications to direct a liposome to the desired target.
  • the method of preparing such a targeting composition having tumour-targeting capacity comprises covalent attachment of a hydrophilic peptide to a synthetic derivative of polyethylene glycol.
  • Another object of this invention is a purification method for the targeting composition obtained by covalently attaching the cyclic GRENYHGCTTHWGFTLC peptide (CTT2 peptide) or a derivative thereof to a synthetic derivative of polyethylene glycol.
  • CTT2 peptide cyclic GRENYHGCTTHWGFTLC peptide
  • the peptide-lipid mixture obtained is incubated with an organic solvent to obtain a precipitate, the precipitate is centrifuged, washed with an organic solvent and recentrifuged to obtain a pellet, the pellet is suspended into a suitable buffer and size-exclusion chromatography is carried out to obtain pure targeting composition.
  • a still further object of this invention is a method for preparing a therapeutic or imaging liposome composition, comprising the steps of obtaining liposomes carrying at least one chemotherapeutic agent or imaging agent, preparing a targeting composition having tumour targeting capacity, by covalently attaching a derivative of small matrix metalloproteinase inhibitor peptide to a synthetic derivative of polyethylene glycol, and combining the liposomes and the targeting composition to form a suspension.
  • Still another object of the invention is a method for treating cancer in a patient, comprising the steps of obtaining liposomes carrying at least one chemotherapeutic agent, obtaining a targeting composition comprising a derivative of small matrix metalloproteinase inhibitor peptide and a synthetic derivative of polyethylene glycol, combining the liposomes and the targeting composition to form a suspension, and administering the suspension obtained to the patient.
  • Still another object of the invention is a diagnostic or imaging composition, comprising a targeting composition comprising a derivative of small matrix metalloproteinase inhibitor peptide and a synthetic derivative of polyethylene glycol, and liposomes carrying at least one imaging agent, or a diagnostic test kit including such a composition.
  • CTT2 peptides were covalently attached to PEG phospholipids through the chemical reaction between the terminal amine of the peptide and the functional NHS (hydroxysuccinimidyl) group at the end of the poly(ethylene glycol) polymer chain of the PEG phospholipid.
  • the reaction between the terminal amine and the active succinimidyl ester of the PEG carboxylic acid produced a stable amide linkage.
  • Different molar ratios of the peptide and the PEG phospholipid, as well as the reaction time and temperature were tested to optimize the coupling reaction.
  • DMF dimethylformamide
  • TAT2 trifluoroacetic acid
  • DSPE-PEG-NHS 3400 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[poly(ethylene glycol) 3400]-N-hydroxysuccinamidyl carbonate
  • DSPE-PEG-NHS 3400 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[poly(ethylene glycol) 3400]-N-hydroxysuccinamidyl carbonate
  • CTT2-PEG-DSPE and CTT2 were extracted from the reaction mixture using diethyl ether ( FIG. 1 ).
  • CTT2-PEG-DSPE was separated from CTT2 using HPLC gel filtration ( FIG. 2 ).
  • the reaction mixture (1 ml) was incubated with 5 ml diethyl ether at ⁇ 20° C. for 1 hour. It was then centrifuged at 13000 rpm for 10 min in a centrifuge that was pre-cooled down to +4° C. The pellet was re-suspended in 5 ml cold diethyl ether and centrifuged again. The pellet was lyophilized for 1 hour.
  • the pellet was dissolved in 100 ⁇ l of 50 mM ammonium acetate buffer +0.1% TFA, pH 4.5, which is the mobile phase in HPLC. Fifty microlitres of the sample were injected at a time. An isocratic run of 1 ml/min was carried out in the AKTA Purifier 10 (Amersham) with the Superdex 75 10/300 GL gel filtration column (Amersham, 1.5 ml) for 1.5 ⁇ column volume. The detection wavelength was 221 nm, with detection at wavelengths 230 and 280 nm for additional information. The fraction(s) containing the product was lyophilized, followed by the re-suspension in 400 ⁇ l of water and lyophilization again in order to remove the ammonium acetate.
  • the amount of the product was measured by a modified version of the Rousell assay as described below. MALDI-TOF analysis was used to confirm the purity and the identity of the product ( FIGS. 3 a ., 3 b . and 3 c .). The integrity of the cyclic structure of the CTT2 peptide was verified by the Ellman's test as described below. For long-term preservation, the lyophilized product can be preserved in dry surroundings at ⁇ 20° C.
  • Each molecule of the product CTT2-PEG-DSPE contains one molecule of phospholipid DSPE. Therefore, by measuring the concentration of the phospholipid DSPE, the concentration of the product is obtained. The phospholipid concentration was measured by a modification of the Rousell assay (Böttcher et al., 1961).
  • the yield of the coupling reaction can be calculated. In average, the coupling yield was around 15%. Therefore, the starting material of one milligram of CTT2 peptide and 2.05 milligrams of DSPE-PEG-NHS would produce approximately 0.5 milligrams of CTT2-PEG-DSPE.
  • DNTB 5,5′-dithio-bis-(2-nitro-benzoic acid) known as DNTB can be used for quantification of free sulfhydryl groups in solution.
  • a solution of this compound produces a quantifiable yellow-coloured product when it reacts with free sulfhydryl groups to yield a mixed disulfide and 2-nitro-5-thiobenzoic acid (TNB).
  • TBN 2-nitro-5-thiobenzoic acid
  • a sulfhydryl group can be quantified by reference to the extinction coefficients of DNTB.
  • Sulfhydryl groups in cyclic peptides are not present, because the cysteines are linked together through S—S bonds.
  • the sulfhydryl groups can be quantified with Ellman's test. This test can be used for making sure that cyclic peptide is still in active form.
  • the test was performed using Ellman's reagent according to the instructions of the manufacturer (Pierce). The results were measured spectrophotometrically at 412 nm. If the value was bigger than 0.020, the peptide was no longer active. Otherwise the cyclic structure of the peptide was still intact. It was shown that the coupling procedure did not disturb the cyclic structure of the CTT2-peptide. However, this test should be performed on each new batch of coupled peptide to validate the quality.
  • CTT2-PEG-DSPE was suspended in 400 ⁇ l of buffer (100 mM histidine, 55 mM sucrose, pH 6.5). To 1 ml Doxil®/Caelyx® solution (Ortho Biotech), 100 ⁇ l of the CTT2-PEG-DSPE micelle suspension was added. The mixture was incubated at +60° C. for 30 min. The suspension was then ready to be injected to mice or humans. The suspension can also be preserved at +4° C. for at least 3 weeks.
  • buffer 100 mM histidine, 55 mM sucrose, pH 6.5
  • Doxil®/Caelyx® solution Ortho Biotech
  • the incorporation efficiency can be measured by using radioisotope-labelled peptide and gel-filtration to separate the unreacted micelle from the liposome.
  • the incorporation efficiency is represented by the percentage of the activity in liposome fractions out of the total activity. Different incubation times and temperatures were tested, and the incubation at +60° C. for 30 min was found to be the optimal reaction conditions. The efficiency of incorporation under these conditions was close to 100%. Based on the average size and surface area of the liposomes, the amount of CTT2 peptide per liposome can be calculated. Under the reaction conditions described above, there are approximately 500 pieces of CTT2 molecules per liposome. Therefore, this amount of CTT2 peptide attached should give the liposome high enough targeting activity.
  • the leakage of doxorubicin from the liposomes after the incorporation experiments at different reaction times and temperatures were determined by comparing the amount of free doxorubicin before and after the experiment. The leakage was found to be minimal (the leakage before the incorporation was in average 4.5% and after the reaction in average 4.2%).
  • A2780 ovarian carcinoma cells were cultured in RPMI 1′medium (Biowhittaker) containing 10% foetal calf serum (Biowhittaker). After harvesting of the cells, 5.0 ⁇ 10 6 cells were injected subcutaneously into posterior flank of 5-6-week-old NMRI nude female mice. The biodistribution study was performed when the tumour size had become about 10 mm in diameter.
  • A2780 ovarian carcinoma-bearing mice were injected with the liposomal doxorubicin dose of 9 mg of doxorubicin/kg via a tail vein.
  • mice were killed 2 h, 6 h, 24 h, 48 h, 72 h and 96 h after the injection for the collection of blood, heart, liver, kidney, lung, muscle, brain, spleen and tumour samples.
  • the blood was centrifuged at 5000 rpm for 10 min at +4° C. to obtain plasma.
  • the tissues were frozen in liquid nitrogen and lyophilized for two days in dark.
  • the dried tissues were weighed and extracted with acid alcohol (0.3M HCl in 50% EtOH) to obtain the final concentration of 20 mg/ml.
  • the tissue homogenates were centrifuged at 13000 ⁇ g for 10 min at +4° C.
  • the cleared plasma and the cleared tissue extracts were determined for doxorubicin fluorescence using spectrofluoro-meter (Varian). Doxorubicin fluorescence was analysed by monitoring the fluorescence intensity at 590 nm using excitation wavelength of 470 nm, and comparing with standard samples containing known amounts of doxorubicin that had been processed in the same manner.
  • CTT2-coated Doxil®/Caelyx (CTT-SL) accumulation in tumour was 46.2% higher than the tumour accumulation of Doxil®/Caelyx® (SL) over a period of 96 hours ( FIG. 4 ). This shows the significant increase in the tumour targeting capacity of CTT2-coated Doxil®/Caelyx®.
  • A2780 cells were injected subcutaneously into the posterior flanks of 50 NMRI nude female mice.
  • the mice were randomly allocated into five treatment groups.
  • the mice were treated with drugs when the tumour size had grown 5 mm in diameter (65 mm 3 ).
  • the mice received three drug injections of 9 mg liposomal or free doxorubicin/kg in three-day intervals.
  • Doxorubicin concentration in CTT2-coated Doxil®/Caelyx (CTT-SL), Doxil®/Caelyx (SL) and free formulations was 2 mg/ml and thus the injection volumes varied between 120-150 l.
  • mice were weighed and their tumour sizes were measured twice a week after treatment initiation. When tumour sizes exceeded 1000 mm 3 the mice were sacrificed.
  • CTT2-PEG-DSPE was produced as described above.
  • ten immunodeficient mice were inoculated with human ovarian carcinoma cells (OV-90).
  • the biodistribution study was performed by injecting iodine-labelled CTT2-PEG-DSPE (200 ⁇ g; ⁇ 1 MBq) in 200 ⁇ l PBS into the tail vein of mice.
  • the mice were sacrificed and their blood and tissues were dissected for gamma counting. Highest accumulation of radioactivity was observed in tumour xenografts at both time points studied (tumour/muscle ratio 43) ( FIG. 6 .).
  • CTT2 can be viewed as having two structurally distinct parts. Cyclic (-CTTHWGFTLC) part of the peptide is more hydrophobic compared to the linear GRENYHG- part of the peptide. The attachment point (N-terminus vs. C-terminus) of CTT2 peptide to any molecular moiety might have effect on conjugate solubility and bioactivity. Two different peptide derivatives (peptides 1 and 4 in Table 1) were synthesized in order to improve the solubility and bioactivity of conjugates.
  • the peptides can be used as probes for in vivo imaging of physiological states and processes.
  • CTT2 peptide can be directly labelled with radioactive iodine.
  • More sophisticated radioactive imaging agents e.g. 111 In and 99m Tc require a chelator moiety conjugated to original peptide.
  • DOTA derivatives of CTT2 peptide (peptides 2, 3 and 5 in Table 1) were synthesized, and one of them (peptide 3 in Table 1) was labelled with cold indium.
  • These peptide-DOTA conjugates (peptides 2 and 5 in Table 1) can be labelled with radioactive isotopes to be used either in diagnostic ( 111 In ) or therapeutic purposes ( 177 Lu, 90 Y).
  • 6F-Trp CTT2 By synthetic incorporation of an unnatural fluorotryptophan amino acid, we obtained two CTT2-peptide derivatives, 6F-Trp CTT2 and 5F-Trp CTT2 (peptides 6 and 7 in Table 1).
  • the 6F-Trp CTT2 showed enhancement in serum stability and improved ability to inhibit tumour cell migration in comparison to the wild type peptide (see Biodistribution of the 6F-Trp CTT2 peptide).
  • a 5-OH-Trp derivative was prepared (peptide 8 in Table 1).
  • the peptides were synthesized with an Applied Biosystems model 433A (Foster City, Calif.) using Fmoc-chemistry as reported previously (Koivunen et al., 1999), except that the disulfide bond formation was conducted using hydrogen peroxide.
  • the peptide was dissolved in 50 mM ammonium acetate (pH 7.5) at a 1 mg/ml concentration and 0.5 ml of 3% hydrogen peroxide per 100 mg peptide was added. After 30 min incubation, pH was adjusted to 3.0 and the cyclized peptide was purified by reverse-phase HPLC using a linear acetonitrile gradient (0%-70% during 30 min) in 0.1% trifluoroacetic acid.
  • Indium labelling of DOTA derived peptide 1.2 mg of K(DOTA)RENYHG-cyclo-(CTTHWGFTLC) was dissolved in 100 ⁇ l of ammonium acetate buffer (pH 6.5). InCl 3 was dissolved in ammonium acetate buffer (pH 6.5). Two molar equivalents of InCl 3 solution were added to the peptide solution. Reaction mixture was left standing overnight at RT. Indium-labelled peptide was purified by reverse phase C-18 cartridges using ammonium acetate buffer (pH 6.5) and acetonitrile solution (50%/50%). Indium-labelled peptides were obtained as white solid after lyophilization of freezed eluates.
  • the 6F-Trp CTT2 peptide was used in biodistribution study to evaluate its kinetic and tumour targeting properties.
  • the study was performed in mice with established human ovarian carcinoma tumours (OV-90).
  • the 6F-Trp CTT2 peptide was labelled with iodine-125.
  • 40 ⁇ g of purified and labelled peptide ( ⁇ 1 MBq) was injected into the tail vein of mice. 30 min and 180 min after peptide injection mice were sacrificed and blood and tissue samples were collected. The accumulated radioactivity was determined with gamma counter.
  • the results showed a remarkable accumulation of radioactivity in tumour tissue with tumour/muscle ratios 14.9 and 23.3 at 30 min and 180 min, respectively. Instead, in all other organs the accumulation of radioactivity was negligible and the clearance was comparable to blood ( FIG. 8 ).
  • the possibility of using unnatural amino acids in peptide synthesis may provide more active and stable peptides for tumour targeting.
US10/576,086 2003-10-17 2004-10-15 Targeting compositions and preparation therof Abandoned US20070140972A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
FI20031528A FI20031528A0 (fi) 2003-10-17 2003-10-17 Terapeuttinen liposomikoostumus ja menetelmä sen valmistamiseksi
FI20031528 2003-10-17
PCT/FI2004/050150 WO2005037862A1 (en) 2003-10-17 2004-10-15 Targeting compositions and preparation thereof

Publications (1)

Publication Number Publication Date
US20070140972A1 true US20070140972A1 (en) 2007-06-21

Family

ID=29225968

Family Applications (1)

Application Number Title Priority Date Filing Date
US10/576,086 Abandoned US20070140972A1 (en) 2003-10-17 2004-10-15 Targeting compositions and preparation therof

Country Status (6)

Country Link
US (1) US20070140972A1 (ja)
EP (1) EP1685153A1 (ja)
JP (1) JP2008505049A (ja)
CA (1) CA2542684A1 (ja)
FI (1) FI20031528A0 (ja)
WO (1) WO2005037862A1 (ja)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090162425A1 (en) * 2007-09-19 2009-06-25 University Of Tennessee Research Foundation Methods and compositions for inhibiting undesirable cellular proliferation by targeted liposome delivery of active agents
US20130121915A1 (en) * 2010-04-05 2013-05-16 Bar-Llan University Protease-activatable pore-forming polypeptides
ITUB20160191A1 (it) * 2016-01-21 2017-07-21 Invectors S R L Kit per la preparazione di doxorubicina liposomiale funzionalizzata con peptidi per il target selettivo di recettori sovra espressi da cellule tumorali

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7261876B2 (en) 2002-03-01 2007-08-28 Bracco International Bv Multivalent constructs for therapeutic and diagnostic applications
US8623822B2 (en) 2002-03-01 2014-01-07 Bracco Suisse Sa KDR and VEGF/KDR binding peptides and their use in diagnosis and therapy
US20050100963A1 (en) 2002-03-01 2005-05-12 Dyax Corporation KDR and VEGF/KDR binding peptides and their use in diagnosis and therapy
US7794693B2 (en) 2002-03-01 2010-09-14 Bracco International B.V. Targeting vector-phospholipid conjugates
FI20040682A0 (fi) * 2004-05-14 2004-05-14 Ctt Cancer Targeting Tech Oy Tuumorien ja mestastaasien kuvantaminen käyttäen gelatinaasiin targetoituvaa peptidiä
FI20050695A0 (fi) * 2005-06-30 2005-06-30 Ctt Cancer Targeting Tech Oy Menetelmä fosfolipidi-PEG-biomolekyyli-konjugaattien valmistamiseksi
MX2008007321A (es) * 2005-12-09 2008-09-30 Bracco Int Bv Conjugados fosfolipido de vector dirigido.
CN101489593B (zh) 2006-06-19 2015-11-25 约翰·霍普金斯大学 通过脂质体酶的治疗剂肿瘤特异性递送
JP2012510477A (ja) 2008-12-02 2012-05-10 ザ ユニバーシティー オブ メルボルン 放射性医用薬剤としての窒素含有大環状結合体
US8871189B2 (en) 2011-11-30 2014-10-28 Mallinckrodt Llc MMP-targeted therapeutic and/or diagnostic nanocarriers
US9457107B2 (en) 2011-12-06 2016-10-04 The University Of Melbourne Cage amine ligands for metallo-radiopharmaceuticals
JP2017098216A (ja) * 2016-06-28 2017-06-01 住友化学株式会社 非水電解液二次電池用絶縁性多孔質層および非水電解液二次電池用積層セパレータ

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040213833A1 (en) * 2001-03-26 2004-10-28 Oula Penate Medina Liposomes targeting of matrix metalloproteinase inhibitors

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FI20021726A0 (fi) * 2002-09-27 2002-09-27 Ctt Cancer Targeting Tech Oy Menetelmä peptidien tuottamiseksi

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040213833A1 (en) * 2001-03-26 2004-10-28 Oula Penate Medina Liposomes targeting of matrix metalloproteinase inhibitors

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090162425A1 (en) * 2007-09-19 2009-06-25 University Of Tennessee Research Foundation Methods and compositions for inhibiting undesirable cellular proliferation by targeted liposome delivery of active agents
US20130121915A1 (en) * 2010-04-05 2013-05-16 Bar-Llan University Protease-activatable pore-forming polypeptides
US9073990B2 (en) * 2010-04-05 2015-07-07 Bar-Llan University Protease-activatable pore-forming polypeptides
ITUB20160191A1 (it) * 2016-01-21 2017-07-21 Invectors S R L Kit per la preparazione di doxorubicina liposomiale funzionalizzata con peptidi per il target selettivo di recettori sovra espressi da cellule tumorali

Also Published As

Publication number Publication date
FI20031528A0 (fi) 2003-10-17
EP1685153A1 (en) 2006-08-02
WO2005037862A1 (en) 2005-04-28
JP2008505049A (ja) 2008-02-21
CA2542684A1 (en) 2005-04-28

Similar Documents

Publication Publication Date Title
Marqués-Gallego et al. Ligation strategies for targeting liposomal nanocarriers
US6224903B1 (en) Polymer-lipid conjugate for fusion of target membranes
US7521419B2 (en) Peptide-based compounds
US20070140972A1 (en) Targeting compositions and preparation therof
US5891468A (en) Fusogenic liposome compositions and method
Dal Corso et al. αVβ3 Integrin-targeted peptide/peptidomimetic-drug conjugates: In-depth analysis of the linker technology
US8404802B2 (en) Peptide-based compounds
KR102051038B1 (ko) 나노입자 약물 컨쥬게이트
Medina et al. Radionuclide imaging of tumor xenografts in mice using a gelatinase-targeting peptide
CN107648618B (zh) 一种药物递送体系及其制备方法与应用
MXPA06001090A (es) Agentes de imagenologia de peptido biciclico.
KR20030061811A (ko) 펩티드-기재 화합물
CN109432432B (zh) 靶向至细胞内质网纳米载药系统的构建与应用
CN105188729B (zh) 用于针对肾毒性活性物质进行保护的缀合物
Bak et al. Affinity induced surface functionalization of liposomes using Cu-free click chemistry
FI113840B (fi) Matriisi-metalloproteinaasi-inhibiittorien käyttö liposomien kohdentamisessa
WO2019126565A1 (en) Lipid derivatives for in vitro or in vivo delivery
Laverman et al. In-depth evaluation of the cycloaddition–retro-Diels–Alder reaction for in vivo targeting with [111In]-DTPA-RGD conjugates
WO2003017938A2 (en) Conjugates targeted to target receptors
EP1590317B1 (de) Enantiomerenreines (4s,8s)- und (4r, 8r)-4-p-nitrobenzyl-8-methyl-3, 6, 9-triaza- sp 3 /sp n, sp 6 /sp n, sp 9 /sp n-tricarboxymethyl-1, 11-undecandisäure und deren abkömmlinge, verfahren zu deren herstellung und verwendung zur herstellung pharmazeutischer mittel
WO2020249745A1 (en) Cationic micelles and methods for using them
JP2018534317A (ja) 結腸がんの処置および検出のための組成物および方法
Cho Self-Assembling Lipopeptide Nanocarriers for Targeted Cellular Uptake
Finbloom Supramolecular chemical biology for applications in protein modification and nanomedicine
CN116622005A (zh) 聚合物前药化合物及其制备方法和聚合物前药胶束及其制备方法

Legal Events

Date Code Title Description
AS Assignment

Owner name: CTT CANCER TARGETING TECHNOLOGIES OY, FINLAND

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:ZHU, YING;VALTANEN, HELI;KAUKINEN, SAMI;AND OTHERS;REEL/FRAME:018893/0939;SIGNING DATES FROM 20060411 TO 20060413

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION